Endothelial receptor-mediated binding of glucose-modified albumin is associated with increased monolayer permeability and modulation of cell surface coagulant properties.

Abstract

Advanced glycosylation end products (AGE) of proteins accumulate in the vasculature with diabetes and aging, and are thought to be associated with vascular complications. This led us to examine the interaction of AGE-BSA as a prototype of this class of nonenzymatically glycosylated proteins subjected to further processing, with endothelium. Incubation of 125I-AGE-BSA with cultured bovine endothelium resulted in time-dependent, saturable binding that was half-maximal at a concentration of approximately 100 nM. Although unlabeled normal BSA was not a competitor, unlabeled AGE-BSA was an effective competitor of 125I-AGE-BSA-endothelial cell interaction. In addition, AGE modification of two alternative proteins, hemoglobin and ribonuclease, rendered them inhibitors of 125I-AGE-BSA binding to endothelium, although the native, unmodified forms of these proteins were not. At 37 degrees C, binding of 125I-AGE-BSA or gold-labeled AGE-BSA was followed by internalization and subsequent segregation either to a lysosomal compartment or to the endothelial-derived matrix after transcytosis. Exposure of endothelium to AGE-BSA led to perturbation of two important endothelial cell homeostatic properties, coagulant and barrier function. AGE-BSA downregulated the anticoagulant endothelial cofactor thrombomodulin, and induced synthesis and cell surface expression of the procoagulant cofactor tissue factor over the same range of concentrations that resulted in occupancy of cell surface AGE-BSA binding sites. In addition, AGE-BSA increased endothelial permeability, resulting in accelerated passage of an inert macromolecular tracer, [3H]inulin, across the monolayer. These results indicate that AGE derivatives of proteins, potentially important constituents of pathologic vascular tissue, bind to specific sites on the endothelial cell surface and modulate central endothelial cell functions. The interaction of AGE-modified proteins with endothelium may play an important role in the early stages of increased vascular permeability, as well as vessel wall-related abnormalities of the coagulation system, characteristic of diabetes and aging.

Toxic action of advanced glycation end products on cultured retinal capillary pericytes and endothelial cells: relevance to diabetic retinopathy

“…In contrast to previous reports we failed to observe the release of any degradation products into the media over the 24-h incubation. The reason for this discrepancy is at present unclear but one explanation could be that degraded material released by the confluent monolayer may become associated with the extracellular matrix components [10]. The finding that the pool of cell surfaceassociated AGE-BSA in BAEC is much lower than the pool of internalized ligand is consistent with the observation made by Schmidt et al [31] with macrophages.…”

“…The finding that AGE-BSA is also toxic to aortic endothelial cells but only at the high concentration of 62.5 m g/ml is in agreement with Tezuka et al [26] showing that AGE-BSA at 50 m g/ml inhibits the proliferation of cultured human umbilical cord vein endothelial cells (HUVEC). Despite the low level of AGE-mediated toxicity in BAEC, it is possible that exposure to AGE-BSA may have resulted in changes to the cytoskeleton and cell permeability as reported by Esposito et al [10]. These changes were not investigated in the present study.…”

“…Previous studies have reported that AGE-modified proteins have a number of biological effects [28], endothelial cell migration and tube formation [26], alteration in cell shape/ cytoskeleton along with perturbation of barrier and coagulant function [10]. Most of these effects are suggested to be mediated by the interaction of AGE with AGE-specific receptors which are localized on various cell types, including smooth muscle cells, monocytes, macrophages and mesangial cells [29].…”

“…The addition of AGE-modified proteins to cultures of mouse mesangial cells results in the increased synthesis and production of extracellular matrix components, such as fibronectin and collagen [9]. AGE-modified proteins bind specifically to AGE-receptors on the surface of endothelial cells, resulting in receptor-mediated endocytosis, changes in cellular proliferation, endothelial cell permeability, coagulant functions [10] and altered expression of endothelialderived relaxing activity [11]. Receptors that specifically recognize AGE adducts on proteins have also been identified on macrophages, mesangial cells and other cell types [12,13].…”

Effect of advanced glycation end products on oxidative stress in endothelial cells in culture: a warning on the use of cells studied in serum-free media

“…In vitro, it was also shown that AGE-BSA caused cytoskeletal rearrangement and increased inulin permeability in a bovine aortic endothelial monolayer preparation [11]. However, the actual AGEs mediating these responses have not been identified, although one recent, but unconfirmed, report [12] suggests that N e -(carboxymethyl)lysine (CML) could be a ligand for RAGE.…”